Methods and Compositions for Preserving the Viability of Photoreceptor Cells

ABSTRACT

Provided are methods and compositions for maintaining the viability of photoreceptor cells following retinal detachment. The viability of photoreceptor cells can be preserved by administering an apoptosis inhibitor to a mammal having an eye with retinal detachment. The apoptosis inhibitor maintains the viability of the photoreceptor cells until such time that the retina becomes reattached to the underlying retinal pigment epithelium and choroid. The treatment minimizes the loss of vision, which otherwise may occur as a result of retinal detachment.

RELATED APPLICATIONS

This application is a continuation-in-part of International ApplicationNo. PCT/US03/01648, filed Jan. 17, 2003, which claims the benefit ofU.S. Provisional Application No. 60/349,918, filed Jan. 18, 2002, theentire disclosures of which are incorporated by reference herein.

FIELD OF THE INVENTION

The invention relates generally to compositions and their use forpreserving the viability of photoreceptor cells following retinaldetachment, and more particularly the invention relates to compositionscomprising an apoptosis inhibitor and their use in maintaining theviability of photoreceptor cells following retinal detachment.

BACKGROUND

The retina is a delicate neural tissue lining the back of the eye thatconverts light stimuli into electric signals for processing by thebrain. Within the eye, the retina is disposed upon underlying retinalpigment epithelium and choroid, which provide the retina with a supplyof blood and nutrients. A common and potentially blinding conditionknown as retinal detachment occurs when the retina becomes disassociatedfrom its underlying retinal pigment epithelium and/or choroid with theaccumulation of fluid in the intervening space. The loss of visualfunction appears to be more pronounced when the retinal detachmentsinvolve the central macula.

Unless treated, retinal detachments often result in irreversible visualdysfunction, which can range from partial to complete blindness. Thevisual dysfunction is believed to result from the death of photoreceptorcells, which can occur during the period when the retina is detachedfrom its underlying blood and nutrient supply. Reattachment of theretina to the back surface of the eye typically is accomplishedsurgically, and despite the good anatomical results of these surgeries(i.e., reattachment of the retina) patients often are still left withpermanent visual dysfunction.

There is still a need for new methods and compositions for maintainingthe viability of photoreceptor cells following retinal detachment andfor preserving vision when the retina ultimately becomes reattached.

SUMMARY

It is understood that photoreceptor cells in the retina may die via avariety of cell death pathways, for example, via apoptotic and necroticcell death pathways. It has been found, however, that upon retinaldetachment, the photoreceptor cells predominantly undergo apoptotic celldeath in the detached portion of the retina. In addition, it has beenfound that, among other things, one or more caspases, for example,caspase 3, caspase 7, caspase 8, and caspase 9, participate in thecascade of events leading to apoptotic cell death.

In one aspect, the invention provides a method of preserving theviability of photoreceptor cells in a mammalian eye following retinaldetachment. More particularly, the invention provides a method ofpreserving the viability of photoreceptor cells disposed within a regionof a retina that has become detached from its underlying retinal pigmentepithelium and/or choroid. The method comprises administering to amammal in need of such treatment an amount of an apoptosis inhibitorsufficient to preserve the viability of photoreceptor cells, forexample, rods and/or cones, disposed within the region of the detachedretina. Administration of the apoptosis inhibitor minimizes the loss ofvisual function resulting from the retinal detachment. The apoptosisinhibitor reduces the number of photoreceptor cells in the region of theretina that, without treatment, would die following retinal detachment.

Useful apoptosis inhibitors include agents capable of modulating, forexample, the receptor mediated pathway and/or the intrinsic pathway.Useful apoptosis inhibitors include agents capable of modulating theactivity of a caspase selected from the group consisting of caspase 3,caspase 7, caspase 8, and caspase 9. Furthermore, it is contemplatedthat, under certain circumstances, it can be advantageous to administeralong with the apoptosis inhibitor, another neuroprotective agent, forexample, another apoptosis inhibitor or a neurotrophic factor.

In another aspect, the invention provides a method of preserving theviability of photoreceptor cells in a mammalian eye following retinaldetachment. More particularly, the invention provides a method ofpreserving the viability of photoreceptor cells disposed within a regionof a retina that has become detached from its underlying retinal pigmentepithelium and/or choroid. The method comprises administering to amammal in need of such treatment an amount of a caspase inhibitor, forexample, a caspase 3 inhibitor, a caspase 7 inhibitor, a caspase 8inhibitor or a caspase 9 inhibitor, or a combination of two or more ofsuch caspase inhibitors, sufficient to preserve the viability ofphotoreceptor cells disposed within the region of the detached retina.

Because photoreceptors die as a result of retinal detachment,administration of the apoptosis inhibitor minimizes or reduces the lossof photoreceptor cell viability until such time the retina becomesreattached to the choroid and an adequate blood and nutrient supply isonce again restored. The apoptosis inhibitor minimizes the level ofphotoreceptor cell death, and maintains photoreceptor cell viabilityprior to reattachment of the detached region of the retina. Undercertain circumstances, however, it may be beneficial to administer theapoptosis inhibitor for a period of time after a retinal detachment hasbeen detected and/or the retina surgically reattached. This period oftime may vary depending on the circumstances and can include, forexample, a period of a week, two weeks, three weeks, a month, threemonths, six months, nine months, a year, and two years, after surgicalreattachment.

The apoptosis inhibitor, for example, a caspase inhibitor, can beadministered, either alone or in combination with a pharmaceuticallyacceptable carrier or excipient, by one or more routes. For example, theapoptosis inhibitor may be administered systemically, for example, viaoral or parenteral routes, for example, via intravascular, intramuscularor subcutaneous routes. Alternatively, the apoptosis inhibitor may beadministered locally, for example, via intraocular, intravitreal,intraorbital, subretinal, or transcleral routes. Furthermore, it iscontemplated that the apoptosis inhibitor, for example, a caspaseinhibitor, may be administered with another type of neuroprotectiveagent, for example, a neurotrophic factor, to maintain viability of thephotoreceptor cells disposed within the detached portion of the retina.The apoptosis inhibitor and the neuroprotective agent may beco-administered either simultaneously or one after the other, forexample, the apoptosis inhibitor is administered after theneuroprotective agent or the neuroprotective agent is administered afterthe apoptosis inhibitor.

It is contemplated that the practice of the invention will be helpful inmaintaining the viability of photoreceptor cells in retinal detachmentsirrespective of how the retinal detachments were caused. For example, itis contemplated that the practice of the method of the invention will behelpful in minimizing visual dysfunction resulting from retinaldetachments caused by one or more of the following: a retinal tear,retinoblastoma, melanoma, diabetic retinopathy, uveitis, choroidalneovascularization, retinal ischemia, pathologic myopia, and trauma.

In another aspect, the invention provides an improved method ofreattaching a detached retina in a mammal, for example, a human. Theimprovement comprises administering, either locally to the eye orsystemically, an apoptosis inhibitor in an amount sufficient to preservethe viability of photoreceptor cells in the eye. The apoptosis inhibitorcan be a caspase inhibitor, for example, a caspase 3 inhibitor, acaspase 7 inhibitor, a caspase 8 inhibitor, or a caspase 9 inhibitor.The method may also comprise co-administering the apoptosis inhibitorwith a neuroprotective agent, for example, a neurotrophic factor.

The foregoing aspects and embodiments of the invention may be more fullyunderstood by reference to the following figures, detailed descriptionand claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The objects and features of the invention may be more fully understoodby reference to the drawings described below in which:

FIG. 1 is a schematic representation of the intrinsic and theFAS-mediated apoptosis pathways, however, for clarity some of theintermediates in each pathway are not shown and the abbreviationsinclude Cyto C cytochrome C, Apaf-1—apoptosis activating factor 1, Casp3—caspase 3, Casp 7—caspase 7, Casp 8—caspase 8, Casp9—caspase 9,tBID—truncated BID, and PARP—poly-ADP ribose-polymerase;

FIG. 2 depicts a bar chart showing the ratio of cleaved caspase 3 topro-caspase 3 in densitometry units in detached retinas (hatched bars)and attached retinas (solid bars) at one, three and five days postretinal detachment;

FIG. 3 depicts a bar chart showing the ratio of cleaved caspase 9 topro-caspase 9 in densitometry units in detached retinas (hatched bars)and attached retinas (solid bars) at one, three and five days postretinal detachment;

FIG. 4 depicts a bar chart showing the level of caspase 7 indensitometry units in detached retinas (hatched bars) and attachedretinas (solid bars) at one, three and five days post retinaldetachment;

FIG. 5 depicts a bar chart showing the ratio of cleaved poly-ADPribose-polymerase (PARP) to pro-PARP in densitometry units in detachedretinas (hatched bars) and attached retinas (solid bars) at one, threeand five days post retinal detachment

FIG. 6 depicts a bar chart showing the kinetics ofFAS-receptor/FAS-ligand complex formation as a function of time afterretinal detachment (the units on the ordinate axis correspond tonormalized densitometry readings of immunoprecipitated complexes);

FIGS. 7a and 7b depict bar charts showing the kinetics of intrinsicpathway activation as measured by caspase 9 activity levels as afunction of time after retinal detachment (FIG. 7a ) and by caspase9/cytochrome C complex formation as a function of time after retinaldetachment (FIG. 7b );

FIG. 8 depicts a bar chart showing the inhibition of caspase 9 activity24 hours after retinal detachment, as measured in vitro, followinginjection of either DMSO solvent alone or DMSO solvent containing thecaspase 9 inhibitor zLEHD.fmk at the time of detachment; and

FIG. 9 depicts a bar chart showing the inhibition of caspase 9 activity24 hours after retinal detachment, as measured in vitro, followinginjection of anti-FAS-receptor (anti-FAS) neutralizing antibody, oranti-FAS-ligand (anti-FASL) neutralizing antibody at the time ofdetachment.

DETAILED DESCRIPTION

During retinal detachment, the entire retina or a portion of the retinabecomes dissociated from the underlying retinal pigment epithelium andchoroid. As a result, the sensitive photoreceptor cells disposed in thedetached portion of the retina become deprived of their normal supply ofblood and nutrients. If untreated, the retina or more particularly thesensitive photoreceptor cells disposed within the retina die causingpartial or even complete blindness. Accordingly, there is an ongoingneed for methods and compositions that preserve the viability ofphotoreceptor cells following retinal detachment. If photoreceptor celldeath can be minimized during retinal detachment, the affectedphotoreceptors likely will survive once the retina is reattached to theunderlying retinal pigment epithelium and choroid, and thephotoreceptors regain their normal blood and nutrient supply.

Retinal detachment can occur for a variety of reasons. The most commonreason for retinal detachment involves retinal tears. Retinaldetachments, however, can also occur because of, for example,retinoblastomas and other ocular tumors (for example, angiomas,melanomas, and lymphomas), diabetic retinopathy, retinal vasculardiseases, uveitis, retinal ischemia and trauma. Furthermore, retinaldetachments can occur as a result of formation of choroidalneovascularization secondary to, for example, the neovascular form ofage-related macular degeneration, pathologic myopia, and ocularhistoplasmosis syndrome. It is understood that the clinical pathologiesof retinal detachments are different from those of degenerative retinaldisorders, for example, retinitis pigmentosa and age-related maculardegeneration. However, the apoptosis inhibitors discussed herein may beuseful in treating retinal detachments that occur secondary to anunderlying degenerative retinal disorder. Accordingly, it iscontemplated that the methods and compositions of the invention may beuseful in minimizing or otherwise reducing photoreceptor cell deathfollowing retinal detachment, irrespective of the cause of thedetachment.

It is understood that photoreceptor cell death during retinaldetachments may occur as a result of either necrotic or apoptotic (alsoknown as programmed cell death) pathways. Both of these pathways arediscussed in detail in, for example, Kerr et al. (1972) BR. J. CANCER26: 239-257, Wyllie et al. (1980) INT. REV. CYTOLOGY 68: 251-306; Walkeret al. (1988) METH. ACHIE. EXP. PATHOL. 13: 18-54 and Oppenheim (1991)ANN. REV. NEUROSCI. 14: 453-501. Apoptosis involves the orderlybreakdown and packaging of cellular components and their subsequentremoval by surrounding structures (Afford & Randhawa (2000) J. CLIN.PATHOL. 53:55-63). In general, apoptosis, also referred to as anapoptotic pathway, does not result in the activation of an inflammatoryresponse. This is in contrast to necrotic cell death, which ischaracterized by the random breakdown of cells in the setting of aninflammatory response. Typically, during necrosis, also known as anecrotic pathway, a catastrophic event, for example, trauma,inflammation, ischemia or infection, typically causes uncontrolled deathof a large group of cells. There are a variety of assays available fordetermining whether cell death is occurring via a necrotic pathway or anapoptotic pathway (see, for example, Cook et al. (1995) INVEST.OPHTHALMOL. VIS. 36:990-996).

Apoptosis involves the activation of a genetically determined cellsuicide program that results in a morphologically distinct form of celldeath characterized by cell shrinkage, nuclear condensation, DNAfragmentation, membrane reorganization and blebbing (Kerr et al. (1972)BR. J. CANCER 26: 239-257). Assays for detecting the presence ofapoptotic pathways include measuring morphologic and biochemicalstigmata associated with cellular breakdown and packaging, such aspyknotic nuclei, apoptotic bodies (vesicles containing degraded cellcomponents) and internucleosomally cleaved DNA. This last feature isspecifically detected by binding and labeling the exposed 3′-OH groupsof the cleaved DNA with the enzyme terminal deoxynucleotidyl transferasein the staining procedure often referred to as the TdT-dUTP TerminalNick End-Labeling (TUNEL) staining procedure. It is believed that, atthe core of this process lies a conserved set of serine proteases,called caspases, which are activated specifically in apoptotic cells.

In general, during retinal detachment as shown in FIG. 1, apoptosis isactivated by one of two main pathways, the receptor-mediated pathway(Walczak & Krammer (2000) EXP. CELL RES. 256: 58-66) and the intrinsic(mitochondrial) pathway (Loeffler & Kraemer (2000) EXP. CELL RES. 256:19-26). The receptor mediated pathway is understood to involve thecomponents of the FAS/FAS-ligand system; the prototypicalreceptor-mediated apoptosis pathway. Both FAS and FAS-ligand are surfacemembrane proteins that belong to the tumor necrosis factor-α superfamilyof proteins (Love (2003) PROG. NEURO. BIOL. PSYCH. 27: 267-82). As shownin FIG. 1, cleaved caspase 8 can either directly activate caspase 3 ordirectly activate BID, a member of the Bcl-2 family of proteins, whichin turn then feeds into the intrinsic pathway by stimulating the releaseof mitochondrial cytochrome C.

In addition to the receptor-mediated pathway, apoptosis can also becomeactivated via an intrinsic pathway. It is understood that the intrinsicpathway does not involve a surface receptor, but rather results from themodification of intracellular pools of proteins. Such modulators includeBID (activated by the FAS-mediated pathway) as well as other members ofthe Bcl-2 family. Environmental or intracellular stressors result inpost-translation modification of these proteins, which then exert theireffect on the mitochondria to release cytochrome C. It is understoodthat the released cytochrome C then binds with apoptosis activatingfactor-1 and caspase 9 to form a complex known as the apoptosome, whichin turn activates more downstream apoptosis reactions. In particular,the apoptosome, can induce the conversion of pro-caspase 9 into activecleaved caspase 9, which itself then induces the conversion ofpro-caspase 3 into active cleaved caspase 3. Activated caspase 3 (eitheractivated by the FAS-mediated pathway or the intrinsic pathway) theninitiates apoptosis optionally via intermediates caspase 7 and PARP.

The invention provides a method of preserving the viability ofphotoreceptor cells in a mammalian, for example, a primate, for example,a human, eye following retinal detachment. More particularly, theinvention provides a method of preserving the viability of photoreceptorcells disposed within a region of a retina, which has become detachedfrom its underlying retinal pigment epithelium and/or choroid. Themethod may be particularly helpful in preventing vision loss when theregion of detachment includes at least a portion of the macula. Themethod comprises administering to a mammal in need of such treatment anamount of an apoptosis inhibitor sufficient to preserve the viability ofphotoreceptor cells disposed within the region of the detached retina.The apoptosis inhibitor is capable of modulating, for example,decreasing, the activity of one or more of caspase 3, caspase 7, caspase8 and caspase 9, and/or preventing or reducing the activation of one ormore of caspase 3, caspase 7, caspase 8, and caspase 9.

As used herein, the term “apoptosis inhibitor” is understood to mean anyagent other than a naturally occurring neurotrophic factor that, whenadministered to a mammal, reduces apoptotic cell death in photoreceptorcells. It is understood that the apoptosis inhibitor excludes certainnaturally occurring neurotrophic factors, including brain-derivedneurotrophic factor, glial cell line-derived neurotrophic factor,neurotrophin, insulin-like growth factor, ciliary neurotrophic factor,fibroblast growth factor (acidic and basic), transforming growth factorα, and transforming growth factor is understood that certain usefulapoptosis inhibitors act by reducing or eliminating the activity of oneor more members of the intrinsic apoptotic pathway and/or theFAS-mediated apoptotic pathway. For example, it is understood that anagent that inactivates or reduces the activity of the FAS-ligand and/orthe FAS-receptor is considered to be an apoptosis inhibitor.Furthermore, it is understood that an agent that either directly orindirectly affects the activity of a particular caspase, for example,caspase 3, caspase 7, caspase 8, and caspase 9, is considered to be anapoptosis inhibitor.

There are approximately fourteen known caspases, and the activation ofthese proteins results in the proteolytic digestion of the cell and itscontents. Each of the members of the caspase family possess anactive-site cysteine and cleave substrates at Asp-Xxx bonds (i.e., afterthe aspartic acid residue). In general, a caspase's substratespecificity typically is determined by the four residues amino-terminalto the cleavage site. Caspases have been subdivided into subfamiliesbased on their substrate specificity, extent of sequence identity andstructural similarities, and include, for example, caspase 1, caspase 2,caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8,caspase 9, caspase 10, caspase 11, caspase 12, caspase 13 and caspase14. Monitoring their activity can be used to assess the level ofon-going apoptosis.

Furthermore, it has been suggested that apoptosis is associated with thegeneration of reactive oxygen species, and that the product of the Bcl-2gene protects cells against apoptosis by inhibiting the generation orthe action of the reactive oxygen species (Hockenbery et al. (1993) CELL75: 241-251, Kane et al. (1993) SCIENCE 262: 1274-1277, Veis et al.(1993) CELL 75: 229-240, Virgili et al. (1998) FREE RADICALS BIOL. MED.24: 93-101). Bcl-2 belongs to a growing family of apoptosis regulatorygene products, which may either be death antagonists (Bcl-2, bcl-x_(L))or death agonists (Bax, Bak) (Kroemer et al. (1997) NAT. MED. 3:614-620). Control of cell death appears to be regulated by theseinteractions and by constitutive activities of the various familymembers (Hockenbery et al. (1993) CELL 75: 241-251). Several apoptoticpathways may coexist in mammalian cells that are preferentiallyactivated in a stimulus-, stage-, context-specific and cell-type manner(Hakem et al. (1998) CELL 94: 339-352). However, it is contemplated thatagents that upregulate the level of the Bcl-2 gene expression or slowdown the rate of breakdown of the Bcl-2 gene product may be useful inthe practice of the invention.

Although photoreceptors may undergo either apoptotic cell death ornecrotic cell death following retinal detachment it is believed that theprimary mechanism of cell death is via apoptosis. Accordingly, apoptosisinhibitors preferably are used in the practice of the invention.

Useful apoptosis inhibitors include, for example, proteins, for example,cytokines, antibodies and antigen binding fragments thereof (forexample, Fab, Fab′, and Fv fragments), genetically engineeredbiosynthetic antibody binding sites, also known in the art as BABS orsFv's. Other useful apoptosis inhibitors include, for example, peptides,for example, an amino acid sequence less than about 25 amino acids inlength, and optionally an amino acid sequence less than 15 amino acidsin length. Peptides useful in the invention comprise, for example,synthetic peptides and derivatives thereof. Other useful apoptosisinhibitors include, for example, deoxyribose nucleic acids (for example,antisense oligonucleotides and aptamers), ribose nucleic acids (forexample, antisense oligonucleotides and aptamers) and peptidyl nucleicacids, which once administered reduce or eliminate expression of certaingenes, for example, caspase genes as in the case of anti-sensemolecules, or can bind to and reduce or eliminate the activity of atarget protein or receptor as in the case of aptamers. Other usefulapoptosis inhibitors include small organic or inorganic molecules thatreduce or eliminate apoptotic activity when administered to the mammal.

One set of apoptosis inhibitors useful in the practice of the inventioninclude caspase inhibitors. Caspase inhibitors include molecules thatinhibit or otherwise reduce the catalytic activity of a target caspasemolecule (for example, a classical competitive or non-competitiveinhibitor of catalytic activity) as well as molecules that prevent theonset or initiation of a caspase mediated apoptotic pathway.

With regard to the inhibitors of catalytic function, it is contemplatedthat useful caspase inhibitors include, on the one hand, broad spectruminhibitors that reduce or eliminate the activity of a plurality ofcaspases or, on the other hand, specific caspase inhibitors that reduceor eliminate the activity of a single caspase. In general, caspaseinhibitors act by binding the active site of a particular caspase enzymeand forming either a reversible or an irreversible linkage to targetcaspase molecule. Caspase inhibitors may include inhibitors of one ormore of caspase 1, caspase 2, caspase 3, caspase 4, caspase 6, caspase7, caspase 8, caspase 9, caspase 10, caspase 11, caspase 12, caspase 13,and caspase 14.

Useful caspase inhibitors include commercially available synthetic(i.e., non naturally occurring) caspase inhibitors. The syntheticcaspase inhibitors may comprise less that 25, optionally less than 15,and optionally less than 10 amino acids or amino acid derivatives.Synthetic caspase inhibitors typically include a peptide recognitionsequence attached to a functional group such as an aldehyde,chloromethylketone, fluoromethylketone, or fluoroacyloxymethylketone.Typically, synthetic caspase inhibitors with an aldehyde functionalgroup reversibly bind to their target caspases, whereas the caspaseinhibitors with the other functional groups tend to bind irreversibly totheir targets. Useful caspase inhibitors, when modeled withMichaelis-Menten kinetics, preferably have a dissociation constant ofthe enzyme-inhibitor complex (K_(i)) lower than 100 μM, preferably lowerthan 50 μM, more preferably lower than 1 μM. The peptide recognitionsequence corresponding to that found in endogenous substrates determinesthe specificity of a particular caspase. For example, peptides with theAc-Tyr-Val-Ala-Asp-aldehyde sequence are potent inhibitors of caspases 1and 4 (K; =10 nM), and are weak inhibitors of caspases 3 and 7 (IC; >50μM). Removal of the tyrosine residue, however, results in a potent butless specific inhibitor. For example, 2-Val-Ala-Asp-fluoromethylketoneinhibits caspases 1 and 4 as well as caspases 3 and 7.

Exemplary synthetic caspase 1 inhibitors, include, for example,Ac-N-Me-Tyr-Val-Ala-Asp-aldehyde, Ac-Trp-Glu-His-Asp-aldehyde,Ac-Tyr-N-Me-Val-Ala-N-Me-Asp-aldehyde, Ac-Tyr-Val-Ala-Asp-Aldehyde,Ac-Tyr-Val-Ala-Asp-chloromethylketone,Ac-Tyr-Val-Ala-Asp-2,6-dimethylbenzoyloxymethylketone,Ac-Tyr-Val-Ala-Asp(OtBu)-aldehyde-dimethyl acetol,Ac-Tyr-Val-Lys-Asp-aldehyde,Ac-Tyr-Val-Lys(biotinyl)-Asp-2,6-dimethylbenzoyloxymethylketone,biotinyl-Tyr-Val-Ala-Asp-chloromethylketone,Boc-Asp(OBzl)-chloromethylketone,ethoxycarbonyl-Ala-Tyr-Val-Ala-Asp-aldehyde (pseudo acid),Z-Asp-2,6-dichlorobenzoyloxymethylketone, Z-Asp(OlBu)-bromomethylketone,Z-Tyr-Val-Ala-Asp-chloromethylketone,Z-Tyr-Val-Ala-DL-Asp-fluoromethlyketone,Z-Val-Ala-DL-Asp-fluoromethylketone, andZ-Val-Ala-DL-Asp(OMe)-fluoromethylketone, all of which can be obtainedfrom Bachem Bioscience Inc., PA. Other exemplary caspase 1 inhibitorsinclude, for example, Z-Val-Ala-Asp-fluoromethylketone,biotin-X-Val-Ala-Asp-fluoromethylketone, Ac-Val-Ala-Asp-aldehyde,Boc-Asp-fluoromethylketone,Ac-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Pro-Tyr-Val-Ala-Asp-aldehyde(SEQ ID NO: 1), biotin-Tyr-Val-Ala-Asp-fluoroacyloxymethylketone,Ac-Tyr-Val-Ala-Asp-acyloxymethylketone, Z-Asp-CH2-DCB,Z-Tyr-Val-Ala-Asp-fluoromethylketone, all of which can be obtained fromCalbiochem, CA.

Exemplary synthetic caspase 2 inhibitors, include, for example,Ac-Val-Asp-Val-Ala-Asp-aldehyde, which can be obtained from BachemBioscience Inc., PA, and Z-Val-Asp-Val-Ala-Asp-fluoromethylketone, whichcan be obtained from Calbiochem, CA.

Exemplary synthetic caspase 3 precursor protease inhibitors include, forexample, Ac-Glu-Ser-Met-Asp-aldehyde (pseudo acid) andAc-Ile-Glu-Thr-Asp-aldehyde (pseudo acid) which can be obtained fromBachem Bioscience Inc., PA. Exemplary synthetic caspase 3 inhibitorsinclude, for example, Ac-Asp-Glu-Val-Asp-aldehyde,Ac-Asp-Met-Gin-Asp-aldehyde, biotinyl-Asp-Glu-Val-Asp-aldehyde,Z-Asp-Glu-Val-Asp-chloromethylketone,Z-Asp(OMe)-Glu(OMe)-Val-DL-Asp(OMe)-fluoromethylketone, andZ-Val-Ala-DL-Asp(OMe)-fluoromethylketone which can be obtained fromBachem Bioscience Inc., PA. Other exemplary caspase 3 inhibitorsinclude, for example,Ac-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Asp-Glu-Val-Asp-aldehyde(SEQ ID NO: 2), biotin-X-Asp-Glu-Val-Asp-fluoromethylketone,Ac-Asp-Glu-Val-Asp-chloromethylketone, which can be obtained fromCalbiochem, CA. Another exemplary caspase 3 inhibitor includes, thecaspase 3 inhibitorN-benzyloxycarbonal-Asp(OMe)-Glu(OMe)-Val-Asp(Ome)-fluoromethyketone(z-Asp-Glu-Val-Asp-fmk), which can be obtained from Enzyme SystemsProducts, CA.

Exemplary synthetic caspase 4 inhibitors include, for example,Ac-Leu-Glu-Val-Asp-aldehyde and Z-Tyr-Val-Ala-DL-Asp-fluoromethylketone,which can be obtained from Bachem Bioscience Inc., PA, andAc-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Leu-Glu-Val-Pro-aldehyde(SEQ ID NO: 3), which can be obtained from Calbiochem, CA.

Exemplary synthetic caspase 5 inhibitors include, for example,Z-Trp-His-Glu-Asp-fluoromethylketone, which can be obtained fromCalbiochem, CA, and Ac-Trp-Glu-His-Asp-aldehyde andZ-Trp-Glu(O-Me)-His-Asp(O-Me) fluoromethylketone, which can be obtainedfrom Sigma Aldrich, Germany.

Exemplary synthetic caspase 6 inhibitors include, for example,Ac-Val-Glu-Ile-Asp-aldehyde, Z-Val-Glu-Ile-Asp-fluoromethylketone, andAc-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Val-Glu-Ile-Asp-aldehyde(SEQ ID NO: 4), which can be obtained from Calbiochem, CA.

Exemplary synthetic caspase 7 inhibitors include, for example,Z-Asp(OMe)-Gln-Met-Asp(OMe) fluoromethylketone,Ac-Asp-Glu-Val-Asp-aldehyde, Biotin-Asp-Glu-Val-Asp-fluoromethylketone,Z-Asp-Glu-Val-Asp-fluoromethylketone,Ac-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Asp-Glu-Val-Asp-aldehyde(SEQ ID NO: 2), which can be obtained from Sigma Aldrich, Germany.

Exemplary synthetic caspase 8 inhibitors include, for example,Ac-Asp-Glu-Val-Asp-aldehyde, Ac-Ile-Glu-Pro-Asp-aldehyde,Ac-Ile-Glu-Thr-Asp-aldehyde, Ac-Trp-Glu-His-Asp-aldehyde andBoc-Ala-Glu-Va-Asp-aldehyde which can be obtained from Bachem BioscienceInc., PA. Other exemplary caspase 8 inhibitors include, for example,Ac-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Ile-Glu-Thr-Asp-aldehyde(SEQ ID NO: 5) and Z-Ile-Glu-Thr-Asp-fluoromethylketone, which can beobtained from Calbiochem, CA.

Exemplary synthetic caspase 9 inhibitors, include, for example,Ac-Asp-Glu-Val-Asp-aldehyde, Ac-Leu-Glu-His-Asp-aldehyde, andAc-Leu-Glu-His-Asp-chloromethylketone which can be obtained from BachemBioscience Inc., PA. Other exemplary caspase 9 inhibitors include, forexample, Z-Leu-Glu-His-Asp-fluoromethylketone andAc-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Leu-Glu-His-Asp-aldehyde(SEQ ID NO:6), which can be obtained from Calbiochem, CA.

Furthermore, it is contemplated that caspase specific antibodies (forexample, monoclonal or polyclonal antibodies, or antigen bindingfragments thereof), for example, an antibody that specifically binds toand reduces the activity of, or inactivates a particular caspase may beuseful in the practice of the invention. For example, an anti-caspase 3antibody, an anti-caspase 7 antibody, an anti-caspase 8 antibody, or ananti-caspase 9 antibody may be useful in the practice of the invention.Additionally, it is contemplated that an anti-caspase aptamer thatspecifically binds and reduces the activity of, or inactivates aparticular caspase, for example, an anti-caspase 3 aptamer, ananti-caspase 7 aptamer, an anti-caspase 8 aptamer, or an anti-caspase 9aptamer may be useful in the practice of the invention.

Alternatively, certain endogenous caspase inhibitors other thannaturally occurring neurotrophic factors can be used to reduce, orinhibit caspase activity. For example, one useful class of endogenouscaspase inhibitor includes proteins known as inhibitors of apoptosisproteins (IAPs) (Deveraux et al. (1998) EMBO J. 17(8): 2215-2223)including bioactive fragments and analogs thereof. One exemplary IAPincludes X-linked inhibitor of apoptosis protein (XIAP), which has beenshown to be a direct and selective inhibitor of caspase-3, caspase-7 andcaspase-9. Another exemplary IAP includes survivin (see, U.S. Pat. No.6,245,523; Papapetropoulos et al. (2000) J. BIOL. CHEM. 275: 9102-9105),including bioactive fragments and analogs thereof. Survivin has beenreported to inhibit caspase-3 and caspase-7 activity.

Furthermore, by way of example, cAMP elevating agents may also serve aseffective apoptosis inhibitors. Exemplary cAMP elevating agents include,for example, 8-(4-chlorophenylthio)-adenosine-3′:5′-cyclic-monophosphate(CPT-cAMP) (Koike (1992) PROG. NEURO-PSYCHOPHARMACOL. BIOL. PSYCHIAT.16: 95-106), forskolin, isobutyl methylxanthine, cholera toxin (Martinet al. (1992) J. NEUROBIOL. 23:1205-1220), and 8-bromo-cAMP, N⁶,O^(2′)-dibutyryl-cAMP and N⁶,O^(2′)dioctanoyl-cAMP (Rydel and Greene(1988) PROC. NAT. ACAD. SCI. USA 85: 1257-1261).

Furthermore, other exemplary apoptosis inhibitors can include, forexample, glutamate inhibitors, for example, NMDA receptor inhibitors(Bamford et al. (2000) EXP. CELL RES. 256: 1-11) such as eliprodil(Kapin et al. (1999) INVEST. OPHTHALMOL. VIS. SCI 40, 1177-82) andMK-801 (Solberg et al. INVEST. OPHTHALMOL. VIS. SCI (1997) 38,1380-1389) and n-acetylated-α-linked-acidic dipeptidase inhibitors, suchas, 2-(phosphonomethyl) pentanedioic acid (2-PMPA) (Harada et al. NEUR.LETT. (2000) 292, 134-36); steroids, for example, hydrocortisone anddexamethasone (see, U.S. Pat. No. 5,840,719; Wenzel et al. (2001)INVEST. OPHTHALMOL. VIS. SCI. 42: 1653-9); nitric oxide synthaseinhibitors (Donovan et al. (2001) J. BIOL. CHEM. 276: 23000-8); serineprotease inhibitors, for example, 3,4-dichloroisocoumarin andN-tosyl-lysine chloromethyl ketone (see, U.S. Pat. No. 6,180,402);cysteine protease inhibitors, for example, N-ethylmaleimide andiodoacetamide; and anti-sense nucleic acid or peptidyl nucleic acidsequences that lower of prevent the expression of one or more of thedeath agonists, for example, the products of the Bax, and Bak genes.

In addition, or in the alternative, it may be useful to inhibitexpression or activity of members of the caspase cascade that areupstream or downstream of caspase 3, caspase 7 and caspase 9. Forexample, it may be useful to inhibit PARP, which is a component of theapoptosis cascade downstream of caspase 7. An exemplary PARP inhibitorincludes 3-aminobenzamide (Weise et al. (2001) CELL DEATH DIFFER.8:801-807). Other examples include inhibitors of the expression oractivity of Apoptosis Activating Factor-1 (Apaf-1) and/or cytochrome C.Apaf-1 and cytochrome C bind the activated form of caspase 9 to producethe apoptosome complex, which is known to propagate the apoptosiscascade. Thus, any protein (for example, antibody), nucleic acid (forexample, aptamer), peptidyl nucleic acid (for example, antisensemolecule) or other molecule that inhibits or interferes with the bindingof caspase 9 to Apaf-1/cytochrome C can serve to inhibit apoptosis.

It is contemplated that the foregoing and other apoptosis inhibitors nowknown or hereafter discovered may be assayed for efficacy in minimizingphotoreceptor cell death following retinal detachment using a variety ofmodel systems. Basic techniques for inducing retinal detachment invarious animal models are known in the art (see, for example, Andersonet al. (1983) INVEST. OPHTHALMOL. VIS. SCI. 24: 906-926; Cook et al.(1995) INVEST. OPHTHALMOL. VIS. SCI. 36: 990-996; Marc et al. (1998)OPHTHALMOL. VIS. SCI. 39: 1694-1702; Mervin et al. (1999) AM. J.OPHTHALMOL. 128: 155-164; Lewis et al. (1999) AM. J. OPHTHALMOL. 128:165-172). Once a suitable animal model has been created (see, Example 1below) an established or putative apoptosis inhibitor can beadministered to an eye at different dosages. The ability of theapoptosis inhibitor and dosage required to maintain cell viability maybe assayed by one or more of (i) tissue histology, (ii) TUNEL staining,which quantifies the number of TUNEL positive cells per section, (iii)electron microscopy, (iv) immunoelectron microscopy to detect the levelof, for example, apoptosis inducing factor (AIF) in the samples, and (v)immunochemical analyses, for example, via Western blotting, to detectthe level of certain caspases in a sample.

The TUNEL technique is particularly useful in observing the level ofapoptosis in photoreceptor cells. By observing the number of TUNELpositive cells in a sample, it is possible to determine whether aparticular apoptosis inhibitor is effective at minimizing or reducingthe level of apoptosis, or eliminating apoptosis in a sample. Forexample, the potency of the apoptosis inhibitor will have an inverserelationship to the number of TUNEL positive cells per sample. Bycomparing the efficacy of a variety of potential apoptosis inhibitorsusing these methods, it is possible to identify apoptosis inhibitorsmost useful in the practice of the invention.

In addition, the apoptosis inhibitor may be co-administered with aneuroprotective agent. As used herein, the term “neuroprotective agent”means any agent that, when administered to a mammal, either alone or incombination with other agents, minimizes or eliminates photoreceptorcell death in a region of the retina that has become detached from theunderlying retinal pigment epithelium and/or choroid. It is contemplatedthat useful neuroprotective agents include, for example, apoptosisinhibitors, for example, caspase inhibitors, and certain neurotrophicfactors that prevent the onset or progression of apoptosis. Morespecifically, useful neuroprotective agents may include, for example, aprotein (for example a growth factor, antibody or an antigen bindingfragment thereof), a peptide (for example, an amino acid sequence lessthan about 25 amino acids in length, and optionally an amino acidsequence less that about 15 amino acids in length), a nucleic acid (forexample, a deoxyribose nucleic acid, ribose nucleic acid, an antisenseoligonucleotide, or an aptamer), a peptidyl nucleic acid (for example,an antisense peptidyl nucleic acid), an organic molecule or an inorganicmolecule, which upon administration minimizes photoreceptor cell deathfollowing retinal detachment.

It is contemplated that useful neuroprotective agents may include one ormore neurotrophic factors. Exemplary neurotrophic factors include, forexample, Brain Derived Growth Factor (Caffe et al. (2001) INVESTOPHTHALMOL. VIS. SCI. 42: 275-82) including bioactive fragments andanalogs thereof; Fibroblast Growth Factor (Bryckaert et al. (1999)ONCOGENE 18: 7584-7593) including bioactive fragments and analogsthereof; Ciliary Neurotrophic Factor including bioactive fragments andanalogs thereof; and Insulin-like Growth Factors, for example, IGF-I andIGF-II (Rukenstein et al. (1991) J. NEUROSCI. 11:2552-2563) includingbioactive fragments and analogs thereof; and cytokine-associatedneurotrophic factors.

Bioactive fragments refer to portions of an intact template protein thathave at least 30%, more preferably at least 70%, and most preferably atleast 90% of the biological activity of the intact proteins. Analogsrefer to species and allelic variants of the intact protein, or aminoacid replacements, insertions or deletions thereof that have at least30%, more preferably at least 70%, and most preferably 90% of thebiological activity of the intact protein.

With reference to the foregoing proteins, the term “analogs” includesvariant sequences that are at least 80% similar or 70% identical, morepreferably at least 90% similar or 80% identical, and most preferably95% similar or 90% identical to at least a portion of one of theexemplary proteins described herein, for example, Brain Derived GrowthFactor. To determine whether a candidate protein has the requisitepercentage similarity or identity to a reference polypeptide, thecandidate amino acid sequence and the reference amino acid sequence arefirst aligned using the dynamic programming algorithm described in Smithand Waterman (1981) J. MOL BIOL. 147:195-197, in combination with theBLOSUM62 substitution matrix described in FIG. 2 of Henikoff andHenikoff (1992), PROC. NAT. ACAD. SCI. USA 89:10915-10919. Anappropriate value for the gap insertion penalty is −12, and anappropriate value for the gap extension penalty is −4. Computer programsperforming alignments using the algorithm of Smith-Waterman and theBLOSUM62 matrix, such as the GCG program suite (Oxford Molecular Group,Oxford, England), are commercially available and widely used by thoseskilled in the art. Once the alignment between the candidate andreference sequence is made, a percent similarity score may becalculated. The individual amino acids of each sequence are comparedsequentially according to their similarity to each other. If the valuein the BLOSUM62 matrix corresponding to the two aligned amino acids iszero or a negative number, the pairwise similarity score is zero;otherwise the pairwise similarity score is 1.0. The raw similarity scoreis the sum of the pairwise similarity scores of the aligned amino acids.The raw score is then normalized by dividing it by the number of aminoacids in the smaller of the candidate or reference sequences. Thenormalized raw score is the percent similarity. Alternatively, tocalculate a percent identity, the aligned amino acids of each sequenceare again compared sequentially. If the amino acids are non-identical,the pairwise identity score is zero; otherwise the pairwise identityscore is 1.0. The raw identity score is the sum of the identical alignedamino acids. The raw score is then normalized by dividing it by thenumber of amino acids in the smaller of the candidate or referencesequences. The normalized raw score is the percent identity. Insertionsand deletions are ignored for the purposes of calculating percentsimilarity and identity. Accordingly, gap penalties are not used in thiscalculation, although they are used in the initial alignment.

Under certain circumstances, it may be advantageous to also administerto the individual undergoing treatment with the apoptosis inhibitor ananti-permeability agent and/or an anti-inflammatory agent so as tominimize photoreceptor cell death. An anti-permeability agent is amolecule that reduces the permeability of normal blood vessels. Examplesof such molecules include molecules that prevent or reduce theexpression of genes encoding, for example, Vascular Endothelial GrowthFactor (VEGF) or an Intercellular Adhesion Molecule (ICAM) (for example,ICAM-1, ICAM-2 or ICAM-3). Exemplary molecules include antisenseoligonucleotides and antisense peptidyl nucleic acids that hybridize invivo to a nucleic acid encoding a VEGF gene, an ICAM gene, or aregulatory element associated therewith. Other suitable molecules bindto and/or reduce the activity of, for example, the VEGF and ICAMmolecules (for example, anti-VEGF and anti-ICAM antibodies and antigenbinding fragments thereof, and anti-VEGF or anti-ICAM aptamers). Othersuitable molecules bind to and prevent ligand binding and/or activationof a cognate receptor, for example, the VEGF receptor or the ICAMreceptor. Such molecules may be administered to the individual in anamount sufficient to reduce the permeability of blood vessels in theeye. An anti-inflammatory agent is a molecule that prevents or reducesan inflammatory response in the eye. Exemplary anti-inflammatory agentsinclude steroids, for example, hydrocortisone, dexamethasone sodiumphosphate, methylpredisolone, and triamcinolone acetonide. Suchmolecules may be administered to the individual in an amount sufficientto reduce or eliminate an inflammatory response in the eye.

As a result, the invention provides an improved method for treating aretinal detachment. The method involves administering an apoptosisinhibitor before and/or during and/or after surgical reattachment of thedetached retina. The apoptosis inhibitor may be administered to themammal from the time the retinal detachment is detected to the time theretina is repaired, for example, via surgical reattachment. It isunderstood, however, that under certain circumstances, it may beadvantageous to administer the apoptosis inhibitor to the mammal evenafter the retina has been surgically repaired. For example, even afterthe surgical reattachment of a detached retina in patients withrhegmatogenous retinal detachments, persistent subretinal fluid mayexist under the fovea as detected by ocular coherence tomography longafter the surgery has been performed (see, Hagimura et al. (2002) AM. J.OPHTHALMOL. 133:516-520). As a result, even after surgical repair theretina may still not be completely reattached to the underlying retinalpigment epithelium and choroid. Furthermore, when retinal detachmentsoccur secondary to another disorder, for example, the neovascular formof age-related macular degeneration and ocular melanomas, it may bebeneficial to administer the neuroprotective agent to the individualwhile the underlying disorder is being treated so as to minimize loss ofphotoreceptor cell viability. Accordingly, in such cases, it may beadvantageous to administer the apoptosis inhibitor to the mammal for oneweek, two weeks, three weeks, one month, three months, six months, ninemonths, one year, two years or more (i) after retinal detachment hasbeen identified, and/or (ii) after surgical reattachment of the retinahas occurred, and/or (iii) after detection of an underlying degenerativedisorder, so as to minimize photoreceptor cell death.

Once the appropriate apoptosis inhibitors have been identified, they maybe administered to the mammal of interest in any one of a wide varietyof ways. It is contemplated that an apoptosis inhibitor, for example, acaspase inhibitor, can be administered either alone or in combinationwith a neuroprotective agent, for example, a neurotrophic agent. It iscontemplated that the efficacy of the treatment may be enhanced byadministering two, three, four or more different agents either togetheror one after the other. Although the best means of administering aparticular apoptosis inhibitor or combination of an apoptosis inhibitorwith another neuroprotective agent may be determined empirically, it iscontemplated that the active molecules may be administered locally orsystemically.

Systemic modes of administration include both oral and parenteralroutes. Parenteral routes include, for example, intravenous,intrarterial, intramuscular, intradermal, subcutaneous, intranasal andintraperitoneal routes. It is contemplated that the apoptosis inhibitorsadministered systemically may be modified or formulated to target theapoptosis inhibitor to the eye. Local modes of administration include,for example, intraocular, intraorbital, subconjuctival, intravitreal,subretinal or transcleral routes. It is noted, however, that localroutes of administration are preferred over systemic routes becausesignificantly smaller amounts of the apoptosis inhibitor can exert aneffect when administered locally (for example, intravitreally) versuswhen administered systemically (for example, intravenously).Furthermore, the local modes of administration can reduce or eliminatethe incidence of potentially toxic side effects that may occur whentherapeutically effective amounts of an apoptosis inhibitor (i.e., anamount of an apoptosis inhibitor sufficient to reduce, minimize oreliminate the death of photoreceptor cells following retinal detachment)are administered systemically.

Administration may be provided as a periodic bolus (for example,intravenously or intravitreally) or as continuous infusion from aninternal reservoir (for example, from an implant disposed at an intra-or extra-ocular location (see, U.S. Pat. Nos. 5,443,505 and 5,766,242))or from an external reservoir (for example, from an intravenous bag).The apoptosis inhibitor may be administered locally, for example, bycontinuous release from a sustained release drug delivery deviceimmobilized to an inner wall of the eye or via targeted transscleralcontrolled release into the choroid (see, for example, PCT/US00/00207,PCT/US02/14279, Ambati et al. (2000) INVEST. OPHTHALMOL. VIS. SCI.41:1181-1185, and Ambati et al. (2000) INVEST. OPHTHALMOL. VIS. SCI.41:1186-1191). A variety of devices suitable for administering anapoptosis inhibitor locally to the inside of the eye are known in theart. See, for example, U.S. Pat. Nos. 6,251,090, 6,299,895, 6,416,777,6,413,540, and 6,375,972, and PCT/US00/28187.

The apoptosis inhibitor also may be administered in a pharmaceuticallyacceptable carrier or vehicle so that administration does not otherwiseadversely affect the recipient's electrolyte and/or volume balance. Thecarrier may comprise, for example, physiologic saline or other buffersystem.

In addition, it is contemplated that the apoptosis inhibitor may beformulated so as to permit release of the apoptosis inhibitor over aprolonged period of time. A release system can include a matrix of abiodegradable material or a material which releases the incorporatedapoptosis inhibitor by diffusion. The apoptosis inhibitor can behomogeneously or heterogeneously distributed within the release system.A variety of release systems may be useful in the practice of theinvention, however, the choice of the appropriate system will dependupon rate of release required by a particular drug regime. Bothnon-degradable and degradable release systems can be used. Suitablerelease systems include polymers and polymeric matrices, non-polymericmatrices, or inorganic and organic excipients and diluents such as, butnot limited to, calcium carbonate and sugar (for example, trehalose).Release systems may be natural or synthetic. However, synthetic releasesystems are preferred because generally they are more reliable, morereproducible and produce more defined release profiles. The releasesystem material can be selected so that apoptosis inhibitor havingdifferent molecular weights are released by diffusion through ordegradation of the material.

Representative synthetic, biodegradable polymers include, for example:polyamides such as poly(amino acids) and poly(peptides); polyesters suchas poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolicacid), and poly(caprolactone); poly(anhydrides); polyorthoesters;polycarbonates; and chemical derivatives thereof (substitutions,additions of chemical groups, for example, alkyl, alkylene,hydroxylations, oxidations, and other modifications routinely made bythose skilled in the art), copolymers and mixtures thereof.Representative synthetic, non-degradable polymers include, for example:polyethers such as poly(ethylene oxide), poly(ethylene glycol), andpoly(tetramethylene oxide); vinyl polymers-polyacrylates andpolymethacrylates such as methyl, ethyl, other alkyl, hydroxyethylmethacrylate, acrylic and methacrylic acids, and others such aspoly(vinyl alcohol), poly(vinyl pyrolidone), and poly(vinyl acetate);poly(urethanes); cellulose and its derivatives such as alkyl,hydroxyalkyl, ethers, esters, nitrocellulose, and various celluloseacetates; polysiloxanes; and any chemical derivatives thereof(substitutions, additions of chemical groups, for example, alkyl,alkylene, hydroxylations, oxidations, and other modifications routinelymade by those skilled in the art), copolymers and mixtures thereof.

One of the primary vehicles currently being developed for the deliveryof ocular pharmacological agents is the poly(lactide-co-glycolide)microsphere for intraocular injection. The microspheres are composed ofa polymer of lactic acid and glycolic acid, which are structured to formhollow spheres. These spheres can be approximately 15-30 μm in diameterand can be loaded with a variety of compounds varying in size fromsimple molecules to high molecular weight proteins such as antibodies.The biocompatibility of these microspheres is well established (see,Sintzel et al. (1996) EUR. J. PHARM. BIOPHARM. 42: 358-372), andmicrospheres have been used to deliver a wide variety of pharmacologicalagents in numerous biological systems. After injection,poly(lactide-co-glycolide) microspheres are hydrolyzed by thesurrounding tissues, which cause the release of the contents of themicrospheres (Thu et al. (2000) NAT. BIOTECH. 18: 52-57). As will beappreciated, the in vivo half-life of a microsphere can be adjusteddepending on the specific needs of the system.

The type and amount of apoptosis inhibitor administered may depend uponvarious factors including, for example, the age, weight, gender, andhealth of the individual to be treated, as well as the type and/orseverity of the retinal detachment to be treated. As with the modes ofadministration, it is contemplated, that the optimal apoptosisinhibitors and dosages of those apoptosis inhibitors may be determinedempirically. The apoptosis inhibitor preferably is administered in anamount and for a time sufficient to permit the survival of at least 25%,more preferably at least 50%, and most preferably at least 75%, of thephotoreceptor cells in the detached region of the retina.

By way of example, protein-, peptide- or nucleic acid-based apoptosisinhibitors can be administered at doses ranging, for example, from about0.001 to about 500 mg/kg, optionally from about 0.01 to about 250 mg/kg,and optionally from about 0.1 to about 100 mg/kg. Nucleic acid-basedapoptosis inhibitors may be administered at doses ranging from about 1to about 20 mg/kg daily. Furthermore, antibodies may be administeredintravenously at doses ranging from about 0.1 to about 5 mg/kg onceevery two to four weeks. With regard to intravitreal administration, theapoptosis inhibitors, for example, antibodies, may be administeredperiodically as boluses in dosages ranging from about 10 μg to about 5mg/eye, and optionally from about 100 μg to about 2 mg/eye. With regardto transcleral administration, the apoptosis inhibitors, may beadministered periodically as boluses in dosages ranging from about 0.1μg to about 1 mg/eye, and optionally from about 0.5 μg to about 0.5mg/eye.

The present invention, therefore, includes the use of a apoptosisinhibitor, for example, a caspase inhibitor, in the preparation of amedicament for treating an ocular condition associated with a retinaldetachment, for example, a loss of vision as a result of photoreceptorcell death in the region of retinal detachment. A composition comprisingone or more apoptosis inhibitors, one agent optionally being a caspaseinhibitor, may be provided for use in the present invention. Theapoptosis inhibitor or agents may be provided in a kit which optionallymay comprise a package insert with instructions for how to treat thepatient with the retinal detachment. For each administration, theapoptosis inhibitor may be provided in unit-dosage or multiple-dosageform. Preferred dosages of the apoptosis inhibitors, however, are asdescribed above.

Throughout the description, where compositions are described as having,including, or comprising specific components, or where processes aredescribed as having, including, or comprising specific process steps, itis contemplated that compositions of the present invention also consistessentially of, or consist of, the recited components, and that theprocesses of the present invention also consist essentially of, orconsist of, the recited processing steps. Further, it should beunderstood that the order of steps or order for performing certainactions are immaterial so long as the invention remains operable.Moreover, two or more steps or actions may be conducted simultaneously.

In light of the foregoing description, the specific non-limitingexamples presented below are for illustrative purposes and are notintended to limit the scope of the invention in any way.

EXAMPLES Example 1 Detection of Caspase Activity Following RetinalDetachment

This example demonstrates that certain caspases, particularly caspases3, 7 and 9, are activated in photoreceptor cells following retinaldetachment. The example also demonstrates that photoreceptor death ismediated by the intrinsic apoptotic pathway following retinaldetachment.

Experimental retinal detachments were created using modifications ofpreviously published protocols (Cook et al. (1995) INVEST. OPHTHALMOL.VIS. SCI. 36(6):990-6; Hisatomi et al. (2001) AM. J. PATH.158(4):1271-8). Briefly, rats were anesthetized using a 50:50 mixture ofketamine (100 mg/ml) and xylazine (20 mg/ml). Pupils were dilated usinga topically applied mixture of phenylephrine (5.0%) and tropicamide(0.8%). A 20 gauge micro-vitreoretinal blade was used to create asclerotomy approximately 2 mm posterior to the limbus. Care was takennot to damage the lens during the sclerotomy procedure. A Glasersubretinal injector (20 gauge shaft with a 32 gauge tip,Becton-Dickinson, Franklin Lakes, N.J.) connected to a syringe filledwith 10 mg/ml of sodium hyaluronate (Healon®, Pharmacia and UpjohnCompany, Kalamazoo, Mich.) then was introduced into the vitreous cavity.The tip of the subretinal injector was used to create a retinotomy inthe peripheral retina, and then the Healon was slowly injected into thesubretinal space to elevate the retina from the underlying retinalpigment epithelium. Retinal detachments were created only in the lefteye (OS) of each animal, with the right eye (OD) serving as the control.In each experimental eye, approximately one half of the retina wasdetached, allowing the attached portion to serve as a further control.

Following creation of the experimental retinal detachment, intraocularpressures were measured before and immediately after retinal detachmentwith a Tono-pen. No differences in intraocular pressures were noted. Theretinal break created by the subretinal injector was confined only tothe site of the injection.

Light microscopic analysis of the detached retinas showed an increase inmorphologic stigmata of apoptosis as a function of time afterdetachment. Eyes then were enucleated one, three, five and seven daysafter creation of the retinal detachment. For light microscopicanalysis, the cornea and lens were removed and the remaining eyecupplaced in a fixative containing 2.5% glutaraldehyde and 2% formaldehydein 0.1M cacodylate buffer (pH 7.4) and stored at 4° C. overnight. Tissuesamples then were post-fixed in 2% osmium tetroxide, dehydrated ingraded ethanol, and embedded in epoxy resin. One-micron sections werestained with 0.5% toluidine blue in 0.1% borate buffer and examined witha Zeiss photomicroscope (Axiophot, Oberkochen, Germany).

At one day after creation of the detachment, pyknosis in the outernuclear layer was confined to the area of the peripheral retinotomy sitethrough which the subretinal injector was introduced. By three days,however, pyknotic nuclei were seen in the whole outer nuclear layer ofthe retina in the area of the detachment. Extrusion of pyknotic nucleifrom the outer nuclear layer into the subretinal space were observed.The remaining layers of the retina appeared morphologically normal. Noinflammatory cells were seen, and there was no apparent disruption ofthe retinal vasculature. Similar changes were seen in sections fromretinas detached for up to one week. No pyknotic nuclei were seen in thearea of the attached retina or in the fellow, non-detached eye. Theamount of outer nuclear layer pyknosis was similar between detachmentsof three-day or one week duration.

Disruption of the photoreceptor outer segments was a prominent featurein the detached retinas. Outer segments of the control eyes and theattached portions of the experimental eyes had an orderly, parallelarrangement. Detachments produced artifactually during tissue processingin these eyes did not alter the photoreceptor morphology. In contrast,the photoreceptor outer segments of detached retinas were severelydisorganized and lost their normal structural organization.Additionally, outer segments in attached areas had similar lengths,whereas the outer segments in detached areas showed variable lengths.

Internucleosomal DNA cleavage in photoreceptor cells was detected viaTUNEL staining. For TUNEL staining, the cornea and lens were not removedafter enucleation, but rather the whole eye was fixated overnight at 4°C. in a phosphate buffered saline solution of 4% paraformaldehydesolution (pH 7.4). Then, a section was removed from the superior aspectof the globe and the remaining eyecup embedded in paraffin and sectionedat a thickness of 6 μm. TUNEL staining was performed on these sectionsusing the TdT-Fragel DNA Fragmentation Detection Kit (Oncogene Sciences,Boston, Mass.) in accordance with the manufacturer's instructions.Reaction signals were amplified using a preformed avidin:biotinylated-enzyme complex (ABC-kit, Vector Laboratories, Burlingame,Calif.). Internucleosomally cleaved DNA fragments were stained withdiaminobenzidine (DAB) (staining indicates TUNEL positive cells) andsections were then counterstained with methylene green.

TUNEL-positive cells were detected at all time points tested (one,three, five and seven days post-detachment). TUNEL-positive staining wasconfined only to the photoreceptor cell layer. Two eyes with retinaldetachments that persisted for two months were monitored. The TUNELassay at two months did not reveal any staining indicating the presenceof internucleosomally cleaved DNA. The prolonged detachment wasassociated with a marked reduction in the thickness of and number ofcell bodies contained in the outer nuclear layer as compared to thenon-detached retina.

Antibodies specific for caspases 3, 7, 9 and PARP were used in Westernblots to probe total retinal protein extracts at various times aftercreation of the retinal detachment. For Western blot analysis, retinasfrom both experimental and control eyes were manually separated from theunderlying retinal pigment epithelium/choroid at days one, three andfive after creation of the retinal detachment. In eyes with retinaldetachments, the experimentally detached portion of the retina wasseparated from the attached portion of the retina and analyzedseparately. Retinas were homogenized and lysed with buffer containing 1mM ethylene diaminetetraacetic acid/ethylene glycol-bis(2-aminoethylethel-N, N, N′, N′-tetraacetic acid/dithiothreitol, 10 mMHEPES pH 7.6, 0.5% (octylphenoxy)polyethoxyethanol (IGEPAL), 42 mMpotassium chloride, 5 mM magnesium chloride, 1 mM phenylmethanesulfonylfluoride and 1 tablet of protease inhibitors per 10 ml buffer (CompleteMini, Roche Diagnostics GmbH, Mannheim, Germany). Samples were incubatedfor 15 minutes on ice, and then centrifuged at 21,000 rpm at 4° C. for30 min. The protein concentration of the supernatant was determinedusing the Bio-Rad D_(C) Protein Assay reagents (Bio-Rad Laboratories,Hercules, Calif.). Proteins were separated via sodium dodecylsulfate-polyacrylamide gel electrophoresis (7.5% and 15% Tris-HCLReady-Gels, Bio-Rad Laboratories), in which 30 μg of total retinalprotein were applied in each lane. The fractionated proteins weretransferred to a PVDF membrane (Immobilon-P, Millipore, Bedford, Mass.).The resulting membrane was blocked with 5% non-fat dry milk in 0.1% TBSTIGEPAL. The blocked membranes then were incubated with antibodiesagainst caspase 7 (1:1,000; Cell Signaling Technology, Beverly, Mass.),caspase 9 (1:1,000; Medical & Biological Laboratories, Naka-ku Nagoya,Japan), cleaved-caspase 3 (1:1,000; Cell Signaling Technology, Beverly,Mass.), caspase 3 (1:2000; Santa Cruz, Santa Cruz, Calif.) or PARP(1:1000; Cell Signaling Technologies, Beverly, Mass.) overnight at 4° C.Bands were detected using the ECL-Plus reagent (Amersham, Pharmacia,Piscataway, N.J.). Membranes were exposed to HyperFilm (Amersham) anddensitometry was preformed using ImageQuant 1.2 software (MolecularDynamics, Inc., Sunnyvale, Calif.). For each eye tested, densitometrylevels were normalized by calculating the ratio of the cleaved-form tothe pro-form of the protein of interest. Pro-caspase 7 levels werenormalized to the densitometry readings from a non-specific banddetected by the secondary IgG. Five eyes were used for each time point,except for the PARP levels for day 5 after detachment for which onlyfour eyes were used. All statistical comparisons were performed using apaired t-test.

The cleaved, or active form of caspase 3 was elevated in the detachedretinas as compared to the attached retinas. The level ofcleaved-caspase 3 increased as a function of time after detachment, witha peak at approximately three days (see, FIG. 2). No cleaved-caspase 3was detected in the control eye or in the attached portion of the retinain the experimental eye.

The ratio of the active to inactive form of caspase 9 also increased asa function of time after creation of the experimental retinal detachment(see, FIG. 3). The peak level of cleaved-caspase 9 was seen at three tofive days after creation of the detachment. The caspase 7 antibody wasable only to detect the pro-form of the protein. There was, however, asignificant difference in the amount of the pro-form detected in theprotein extract from the detached retinas as compared to the attachedretinas (see, FIG. 4). Western blotting with antibodies against PARP (acomponent of the apoptosis cascade downstream of caspase 7) detected anincrease in the level of cleaved-PARP that was maximal at five daysafter detachment (see, FIG. 5). P-values for the comparisons betweendetached and attached retinas are shown in FIGS. 2-5.

The results demonstrate that caspase 3, caspase 7 and caspase 9 are allactivated in photoreceptor cells following retinal attachment.

Example 2 Activation of FAS-Mediated Apoptotic Pathway in the RetinaFollowing Retinal Detachment

The purpose of this example was to determine whether only the intrinsicpathway becomes activated during retinal detachment, or whether thereceptor-mediated pathway also contributes to photoreceptor death.

Experimental retinal detachments were created in Brown-Norway rats byinjecting 10% hyaluronic acid into the subretinal space. Retinal tissuewas harvested at 2, 4, 8, 24, 72 and 168 hours after creation of thedetachment. Immunoprecipitation was performed to assess forFAS-receptor/FAS-ligand complex formation, and activation of caspase 8and BID was assessed by Western blot analysis. Caspase 9 activity assayand immunoprecipitation of the caspase 9/cytochrome C complex wasperformed at these same time points. The results demonstrate that theFAS-mediated apoptotic pathway is activated during retinal detachment,and that FAS pathway activation precedes that of intrinsic pathway.

2.1. Animal Model

The experiments described in Examples 2-4 were performed in accordancewith the ARVO Statement for the Use of Animals in Ophthalmic and VisionResearch and the guidelines established by the University Committee onUse and Care of Animals of the University of Michigan. Retinaldetachments were created in adult male Brown-Norway rats (300-400 gm)essentially as described in Example 1 but with minor modifications.

Briefly, rats were anesthetized with a 50:50 mix of ketamine (100 mg/ml)and xylazine (20 mg/ml), and pupils were dilated with topicalphenylephrine (2.5%) and tropicamide (1%). A sclerotomy was createdapproximately 2 mm posterior to the limbus with a 20-guagemicrovitreoretinal blade (Walcott Scientific, Marmora, N.J.), withspecial caution to not damage the lens. A Glaser subretinal injector(32-gauge tip; BD Ophthalmic Systems, Sarasota, Fla.) connected to asyringe filled with 10 mg/ml sodium hyaluronate (Healon®; Pharmacia andUpjohn Co., Kalamazoo, Mich.) was introduced through the sclerotomy intothe vitreous cavity. The tip of the subretinal injector was introducedinto the subretinal space through a peripheral retinotomy, and thesodium hyaluronate was slowly injected. The neurosensory retina was thusdetached from the underlying retinal pigment epithelium. In allexperiments, approximately one-third to one-half of the retina wasdetached. Detachments were made in the left eye, with the right eyeserving as the control. For control eyes, a sham surgery was performedin which all components of the procedure were performed exceptintroduction of the subretinal injector and creation of the detachment.In experimental eyes, only the detached portion of the retina washarvested for analysis.

2.2. Western Blot Analysis

Retinas from experimental and control eyes were dissected from theRPE-choroid at 3 and 7 days after retinal detachment. Retinas werehomogenized and lysed with buffer containing 10 mM HEPES (pH 7.6), 0.5%IGEPAL, 42 mM KCL, 1 mM PMSF, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 5 mMMgCL₂, and 1 tablet of protease inhibitors per 10 mL buffer (CompleteMini; Roche Diagnostics GmbH, Mannheim, Germany). The homogenates wereincubated on ice and centrifuged at 22,000 g at 4° C. for 60 minutes.The protein concentration of the supernatant was determined using the DcProtein Assay kit (Bio-Rad Laboratories; Hercules Calif.). The proteinsamples were loaded and run on SDS-Polyacrylamide gels (4-20% Tris-HCLready gels, Bio-Rad Laboratories). After electrophoretic separation theproteins were transferred onto polyvinylidene fluoride (PVDF) membranes(Immobilon-P). Protein bands were visualized with Ponceau S staining andthe lanes assessed for equal loading by densitometry on a non-specificband present across all lanes. Membranes were then placed in 5% nonfatpowdered milk in TBS (150 mM NaCl, 50 mM Tris; pH 7.6) and incubatedovernight at 4° C. on a shaker. Membranes were then incubated with theprimary antibody in 2.5% powdered milk in TBS for overnight at 4° C.Membranes were washed extensively with TBS-T (0.1% Tween 20), and thenincubated with horseradish peroxidase labeled secondary antibody(1:3000, Santa Cruz Biotechnology) for 1 hour at room temperature. Bandswere visualized with ECL-Plus (Amersham, Piscataway, N.J.) according tothe manufacturer's instructions. Antibodies against the followingproteins were used: caspase-8 (1:800 dilution, Santa Cruz Biotechnology,Santa Cruz, Calif.), caspase-9 (1:2000 dilution, MBL, Nakaku, Japan),cytochrome C (1:1000 dilution, BD Biosciences, San Jose, Calif.), BID(1:1000 dilution, Santa Cruz Biotechnology), FAS (1:1000 dilution, SantaCruz Biotechnology), and FAS-ligand (1:2000 dilution, MBL).

2.3. Immunoprecipitation

Retinal samples were isolated as described in Section 2.2. For eachcondition tested, 20 of protein extract was placed in 100 μl ofimmunoprecipitation buffer-A (IP-A)+PMSF (20 mM Tris pH 7.5, 100 mMNaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride) and 100 μl of IP-Bbuffer (100 mM Tris pH 7.5, 100 mM NaCl, 0.4% Triton X-100). Sampleswere first incubated overnight with an anti-FAS antibody (0.2 μganti-FAS rabbit polyclonal IgG (Santa Cruz, sc-716)) at 4° C. withgentle rocking, then incubated for 2 hours in 35 μl of 50% suspension ofprotein G sepharose beads at 4° C. with gentle rocking. Beads wereprewashed 4 times with 1 ml of cold IP-C buffer (50 mM tris pH 7.5, 100mM NaCl, 0.2% Triton X-100), then pelleted at 2200 rpm for 6 minutes.Resuspended beads with attached proteins were diluted with Laemmli Dyeloading buffer and heated at 95° C. for 10 minutes prior to running on a4-20% SDS-PAGE ready gel (Bio-Rad). Western blot analysis was performedas described above using a monoclonal antibody against FAS-Ligand (MBL,D057-3). Immunoprecipitation of the caspase 9/cytochrome C complex wasperformed using a similar protocol, except the antibodies used wereanti-caspase 9 (rabbit polyclonal IgG (Santa Cruz, sc-7885)) and amonoclonal antibody against cytochrome C (MBL, BV-3026-3). Densitometryof Western blot bands was performed using a Kodak 440CF Image Station(Kodak Company, Rochester, N.Y.). For each time point, the densitometryreading of the detached retina was normalized against the densitometryreading of attached retina at the same time point.

2.4. Caspase 9 Activity Assay

Caspase 9 activity was measured using a colorimetric tetrapeptideLEHD-pNA cleavage assay kit according to the manufacturer's instructions(BioVision, Mountain View, Calif.). In this assay, 100 μg of totalretinal protein from either attached or detached retinas were incubatedwith substrate (LEHD-pNA, 200 μM final concentration) at 37° C. for 60min. Absorbance was measured at 405 nm in a microplate reader(SpectraMAX 190, Molecular Devices). As a negative control, retinalprotein was incubated with assay buffer lacking any tetrapeptide. Asecond negative control was used in which assay buffer alone wasincubated with the tetrapeptide. As a positive control, purified caspase9 was incubated with the tetrapeptide alone. For each time point, thecaspase 9 activity in the detached retina was normalized against thecaspase 9 activity in attached retina at the same time point.

2.5. Results

The initial experiment determined whether or not the FAS pathway becomesactivated upon retinal detachment. Immunoprecipitation studiesdemonstrated that the receptor/FAS-ligand complex is formed upon retinaldetachment (data not shown). Activation of caspase 8 and BID wasdemonstrated on Western blot analysis by formation of their cleavedforms, as would be expected by the formation of functionalFAS-receptor/FAS-ligand complex. The peak of FAS-receptor/FAS-ligandcomplex formation occurred 8 hours after retinal detachment (see, FIG.6). This preceded the peak of caspase 9 activity, which occurred 24hours after creation of the detachment (see, FIG. 7a ), whichcorresponded to the peak of caspase 9/cytochrome C complex formation(see, FIG. 7b ). Normalizing the densitometry readings for any decreasein outer nuclear layer thickness that might result from the retinaldetachment did not significantly alter the relative values shown.

These experiments demonstrate that retinal detachment up regulates andactivates the FAS/FAS-ligand pathway. This up regulation occurs at thetranscription level, as demonstrated by the increased levels ofmessenger RNA (data not shown). These components are not just present atincreased levels of pro-form, but become activated by the detachment asevidenced by their cleavage into enzymatically active states. The dataalso shows that FAS activation precedes that of the intrinsic pathway,when taken in conjunction with the ability to decrease the latter'sactivity by inhibition of the former suggests a direct linkage ofactivation between the two.

Example 3 Modulation of Caspase 9 and FAS Receptor Activity FollowingRetinal Detachment

This Example demonstrates that it is possible to module the activity ofcaspase 9 in vivo following retinal detachment. Direct inhibition of theintrinsic pathway was performed using the caspase 9 inhibitorz-Leu-Glu-His-Asp-fluoromethylketone (zLEHD.fmk). Indirect inhibition(via inhibition of FAS complex formation) was performed usingneutralizing antibodies against either the FAS-receptor or FAS-ligand.Injection of zLEHD.fmk into the subretinal space of a detached retinaresulted in decreased caspase 9 activity, as did injection ofanti-FAS-receptor antibody into either the subretinal space orintravitreally.

In these experiments the retina was detached with sodium hyaluronateaccording to the protocol described in Example 2, followed immediatelyby the injection of 5 μl of inhibitor. In one experiment, the directinhibitor of caspase 9-zLEHD.fmk was tested. Five microliters of thezLEHD.fmk (2 mM solution in DMSO) (BioVision) was injected into thesubretinal space of the detached retina using a Hamilton Syringe(Hamilton Corp, Reno, Nev.). Five microliters of DMSO was injected intothe subretinal space of the detached retinas as a control for thesolvent in which the zLEHD.fmk was dissolved. In another experiment, theneutralizing antibody against the FAS-receptor (5 μg in phosphatebuffered saline) (clone ZB4, Upstate, Lake Placid, N.Y.) or FAS-ligand(5 μg in phosphate buffered saline) (clone NOK-1, BD-Biosciences) wasinjected either into the subretinal space or the vitreous cavity.

In all inhibition experiments, the retinas were harvested at 24 hoursafter detachment, as this was the peak of caspase 9 activity seen afterdetachment (as shown in Example 2). The caspase 9 activity in thedetached retina was normalized against the caspase 9 activity inattached retina at the same time point.

Caspase 9 activity levels were used as a measurement of intrinsicpathway activation. The activity levels were tested 24 hours after theretinal detachment was created and inhibitor applied, as this was thetime of peak caspase 9 activity (FIG. 7a ). Injection of the caspase 9inhibitor zLEHD.fmk into the subretinal space of a detached retinasignificantly reduced caspase 9 activity to approximately 50% of thecontrol level (p=0.05) (FIG. 8).

Injection of neutralizing antibodies against either the FAS-receptor orthe FAS-ligand into the subretinal space of the detached retina alsoresulted in the reduction of caspase 9 activity by approximately 50%(p=0.05) (FIG. 9). The effect of intravitreal injection of theseantibodies was less than that seen with a subretinal injection, and didnot reach statistical significance. Intravitreally injectedanti-FAS-receptor antibody reduced caspase 9 activity by only about 30%(p=0.13). Intravitreal injection of anti-FAS-ligand antibody resulted inonly a 10% reduction of caspase 9 activity (p=0.54).

Example 4 Preservation of Photoreceptor Viability Following RetinalDetachment

This example demonstrates that administration of an apoptosis inhibitorcan preserve photoreceptor cells following retinal detachment. Theadministration of a caspase 9 inhibitor reduced the number of apoptoticcells following retinal detachment.

Briefly, the retinal detachments were created in the left eyes of threeBrown Norway rats, as described in Example 2, section 2.1. Thedetachment was located on the temporal portion of the retina, andcomprised approximately one third of the total retinal area.

A first rat received the retinal detachment only. A second rat receivedthe retinal detachment and a caspase 9 inhibitor in DMSO. Briefly,immediately after the retina was detached, 5 μl of the zLEHD.fmk (2 mMsolution in DMSO) (BioVision) was injected into the subretinal space ofthe detached retina using a Hamilton Syringe (Hamilton Corp, Reno,Nev.). A third rat received the retinal detachment and DMSO (solventcontrol). Briefly, 5 μl of DMSO was injected into the subretinal spaceof the detached retinas as a control for the solvent in which thezLEHD.fmk was dissolved.

The rats were allowed to recover from the surgery and were returned totheir cages, as per standard animal care protocols. Seventy-two hours (3days) after creation of the detachments, the eyes were enucleated andimmersion-fixed in 4% paraformaldehyde solution for 24 hours. The fixedeyes were then embedded in paraffin and sectioned for histologicanalysis. TUNEL staining was performed on the sections using acommercially-available kit (TdT-Fragel DNA Fragmentation Detection Kit:Oncogene, Boston, Mass.) according to the manufacturer's instructions.

The number of TUNEL-positive cells/100 cells in the outer nuclear layerwere counted for 3 high power fields per section for 2 separate slides.The results are summarized in Table 1.

TABLE 1 Sample % TUNEL positive cells Attached retina (right eye)  1.3%TUNEL positive Detached retina (left eye) 21.6% TUNEL positive Detachedretina (left eye) 33.3% TUNEL positive plus DMSO only Detached retina(left eye)  4.6% TUNEL positive plus caspase 9 inhibitor

The results in Table 1 demonstrate that in the eyes with the detachedretinas, the administration of the caspase 9 inhibitor significantlyreduced the percentage of apoptotic cells and, therefore, preservedphotoreceptor viability following retinal detachment.

INCORPORATION BY REFERENCE

The entire disclosure of each of the patent and non-patent documentsdisclosed herein is expressly incorporated herein by reference for allpurposes.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes which come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

1. A method of preserving the viability of photoreceptor cells disposed within a retina of a mammalian eye following retinal detachment, the method comprising: administering to a mammal having an eye in which a region of the retina has been detached an amount of an apoptosis inhibitor sufficient to preserve the viability of photoreceptor cells disposed within the region of the detached retina.
 2. The method of claim 1, wherein the apoptosis inhibitor is administered to the mammal prior to reattachment of the region of detached retina.
 3. The method of claim 1, wherein the apoptosis inhibitor is administered to the mammal after reattachment of the region of detached retina.
 4. The method of claim 1, wherein the apoptosis inhibitor is administered locally or systemically.
 5. The method of claim 1, wherein a plurality of apoptosis inhibitors are administered to the mammal.
 6. The method of claim 4, wherein at least one apoptosis inhibitor is administered by intraocular, intravitreal, or transcleral administration.
 7. The method of claim 1, wherein the apoptosis inhibitor reduces the number of photoreceptor cells in the region that die following retinal detachment relative to the number of photoreceptor cells that die in the absence of the apoptosis inhibitor.
 8. The method of claim 1, wherein, prior to administration of the apoptosis inhibitor, the photoreceptor cells undergo apoptotic cell death in the region following retinal detachment.
 9. The method of claim 1, wherein the apoptosis inhibitor is capable of modulating the activity of a caspase selected from the group consisting of caspase 3, caspase 7, caspase 8, and caspase
 9. 10. The method of claim 1, wherein the apoptosis inhibitor is a caspase inhibitor.
 11. The method of claim 1, wherein the apoptosis inhibitor modulates FAS receptor activity.
 12. The method of claim 11, wherein the apoptosis inhibitor is an anti-FAS antibody or an anti-FAS receptor antibody.
 13. The method of claim 1, wherein the method further comprises administering a neuroprotective agent.
 14. The method of claim 1, wherein the photoreceptor cells comprise rods and cones.
 15. The method of claim 1, wherein the retinal detachment occurs as a result of a retinal tear, retinoblastoma, melanoma, diabetic retinopathy, uveitis, choroidal neovascularization, retinal ischemia, pathologic myopia, or trauma.
 16. A method of preserving the viability of photoreceptor cells disposed within a retina of a mammalian eye following retinal detachment, the method comprising: administering to a mammal having an eye in which a region of the retina has been detached an amount of a caspase inhibitor sufficient to preserve the viability of photoreceptor cells disposed within the region of the detached retina.
 17. The method of claim 16, wherein the caspase inhibitor is administered to the mammal prior to reattachment of the region of detached retina.
 18. The method of claim 16, wherein the caspase inhibitor is administered to the mammal after reattachment of the region of detached retina.
 19. The method of claim 16, wherein the caspase inhibitor is administered locally or systemically.
 20. The method of claim 16, wherein the caspase inhibitor is administered by intraocular, intravitreal, subretinal, or transcleral administration. 21-29. (canceled) 